Small interfering RNAs that target NR1 and ERK2 in the spinal cord block inflammatory pain signaling

Inflammatory pain is mediated by receptors and signaling proteins in the spinal cord dorsal horn (SCDH). In Chapter I, I tested the feasibility of using viral vector-derived small interfering RNA (siRNA) to target the expression the NR1 subunit of the NMDA receptor. An in vitro luciferase assay was used to identify active and control (inactive) siRNAs that are expressed by a recombinant adeno-associated virus (rAAV) plasmid. rAAV vectors were then prepared and injected into the mouse lumbar SCDH. Three weeks later, we observed a 60 to 75% knockdown of NR1 mRNA and protein in the targeted area. This knockdown of NR1 expression in SCDH did not alter responses to acute thermal or mechanical stimuli. However, the vector-derived siRNA prevented the mechanical allodynia that occurs at 24 and 48 hr after hindpaw injection of an inflammatory agent, Complete Freund's Adjuvant (CFA). Results from Chapter I demonstrate that vector-derived siRNAs can be used to produce a spatial knockdown of the expression and function of a gene that is confined to the ipsilateral SCDH. In Chapter II, I extended the rAAV-derived siRNA approach to target the expression of extracellular signal-regulated kinase ERK2. ERK1 and ERK2 in the SCDH have been implicated in regulating neuroplasticity that contributes to injury-induced pain. However, the species of ERK or the cell types expressing ERK that are essential to injury-induced pain are unknown. Here, using a neurotropic serotype-2 rAAV that expresses a siRNA specific for ERK2, we selectively inhibited the expression and phosphorylation of ERK2 but not ERK1 in the lumbar SCDH neurons of adult mice. This neuron-restricted knockdown dramatically reduced pain hypersensitivity resulting from the intraplantar injection of CFA at 24, 48 and 96 hours but did not alter baseline heat or mechanical paw withdrawal thresholds, or motor function. Results from Chapter 1I demonstrate a critical role of neuronal ERK2 in injury-induced pain. In addition, vector-derived siRNAs may have therapeutic potential for the management of injury-induced pain resulting from the activation of NMDA receptors and/or neuronal ERK2 in the SCDH.